Protein folding is a major topic of current interest. In these studies it is useful to have probes of local environment within the protein and techniques to determine distances between regions of the protein. We propose to test and calibrate two EPR-based techniques using bovine and human carbonic anhydrase as model systems. 1) The effect of a rapidly relaxing Co(II) ion bound at the active site of the protein on the electron spin-lattice relaxation rate of a nitroxyl spin label attached to an inhibitor or to a cysteine residue of the protein will be used to determine the distance between the metal binding site and the location of the spin label. 2) The effect of neighboring methyl groups on the spin echo dephasing rate for a nitroxyl spin label will be used to characterize the environment of the spin label. Both techniques will be tested with spin labels at known locations in normally folded carbonic anhydrase. These techniques will then be used to characterize the folding intermediate and unfolded state that are observed when guanidine hydrochloride is added to human carbonic anhydrase II. It is anticipated that these techniques will be useful in studies of other proteins to which the spin labels can be attached.